The recent advances in cancer immunotherapy have emphasized the importance of the T cell immune response. In particular, immune checkpoint blockade therapy with anti-PD-L1/PD-1 and anti-CTLA-4 has proven successful in treating a subset of patients and cancers by non-specifically activating T cell responses. To understand how cancer immunotherapy fails in the remaining patient population, we must observe how the T cell immune response develops with treatment. Now with novel technologies that allow for high-throughput single-T cell phenotyping and receptor sequencing and a yeast-display method for ligand identification, we can understand how the natural T cell response behaves in cancer and how it is manipulated by immune checkpoint blockade. The previously undiscovered linkage between T cell phenotype and receptor specificity in cancer will distinguish what makes a productive T cell immune response. Questions to be addressed include 1) what T cell immune responses form against cancer, 2) how are T cell phenotypes altered by immune checkpoint blockade therapy, 3) how do T cell receptor specificities change with therapy, and 4) how can manipulation of antigen-specific responses synergize with cancer immunotherapy. The aggressive B16F10 melanoma tumor model implanted into syngeneic C57BL/6 mice will serve as the system to understand the linkage between phenotypic T cell responses and receptor specificities in the context of immune checkpoint blockade. To address these questions, the experimental plan is to 1) characterize T cell immune responses in B16F10 melanoma by T cell phenotype and receptor repertoire, 2) identify T cell receptor peptide specificities by using yeast-display of C57BL/6 peptide-major histocompatibility complex alleles and predictive statistical models, and 3) provide antigen-specific stimulation of T cell responses in synergistic combination with immune checkpoint blockade therapy. This will be the first study to link individual T cell phenotypic responses and receptor specificities to inform fundamental changes in immune responses by immune checkpoint blockade. The discoveries will guide future design of cancer immunotherapeutic strategies and provide new cancer targets when applied to clinical settings.